Exogenous melatonin enhanced cadmium stress tolerance of cucumber seedlings (Cucumis sativus L.)

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Xin Kang, Zi-Qi Pei, Ting-Ting Xu, Cui-Yun Dong, Xue Bai, Juan Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3365346/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Mar, 2024 Read the published version in Biologia → Version 1 posted 5 You are reading this latest preprint version Abstract Melatonin (MT) is a phytohormone that can improve plant stress resistance by regulating physiological processes and gene expression. The present study investigated the role of exogenous MT in alleviating cadmium (Cd) stress in cucumber seedlings. The results showed that Cd stress inhibited the growth of cucumber seedlings and exogenous MT reversed adverse effects of Cd stress. Compared with Cd treatment, MT + Cd treatment enhanced antioxidant enzyme activities, suppressed ROS production and improved photosynthesis in cucumber seedlings. Further research showed that hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO) played important roles in MT enhanced Cd stress tolerance in cucumber seedlings. RNA-seq results indicated that MT was not only related to photosynthetic and antioxidant systems in alleviating Cd injury in cucumber seedlings, but also various phytohormones, heavy metal transporter proteins and transcription factors were also involved. In addition, we selected six differentially expressed genes for qRT-PCR validation, the verification results were consistent with the RNA-seq results. In summary, exogenous MT pretreatment can alleviate Cd toxicity by enhancing antioxidant defense capacity and photosynthetic efficiency of cucumber seedlings, both H 2 O 2 and NO play important roles. In addition, various phytohormones, transcription factors and heavy metal transport proteins are also involved in this regulation of MT. Cucumber Melatonin Cadmium stress RNA-seq Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 1. Introduction In recent years, with the rapid development of industry and the excessive use of agricultural chemicals, the problem of heavy metal pollution of soil has become increasingly serious (Haider et al., 2021 ). As a non-essential metal element for plant growth and development, cadmium (Cd) is easily absorbed by plants. Excessive accumulation of Cd 2+ can injure the structure and function of the plant membrane system and cause oxidative damage, which in reverse inhibits plant development and growth (Arif et al., 2019 ; He et al., 2017 ; Huybrechts et al., 2019 ). In addition, the accumulation of large amounts of Cd 2+ in plants can cause a variety of toxic symptoms, such as leaf yellowing, photosynthetic inhibition, root elongation inhibition, root tip browning, and biomass reduction, which can even lead to the death of the plant (Fan et al., 2023 ; Guo et al., 2022 ; Mohamed et al., 2012 ). Melatonin (MT) is a multifunctional molecule that is ubiquitously found in various plants. Numerous literatures have reported that MT not only promotes plant growth and development (Arnao and Hernández-Ruiz, 2015 ; Teng et al., 2022 ; Wang et al., 2022b ) but it also plays an important role in enhancing abiotic stress tolerance in plants (Shi et al., 2016 ; Zhao et al., 2022 ). For example, Xiao et al. ( 2018 ) showed that low concentrations of MT could promote cotton seed germination by regulating hormone levels and antioxidant enzyme activities. Meanwhile, exogenous MT can also promote the elongation of Arabidopsis primary roots in an indole acetic acid (IAA)-dependent manner (Yang et al., 2021 ). When cucumber is suffering from low-temperature stress, exogenous MT can enhance the tolerance of cucumber seedlings to low-temperature stress by modulating its antioxidant system and photosynthesis(Zhang et al., 2021a ). Under salt stress, exogenous MT can enhance salt tolerance of maize seedlings by increasing their photosynthetic efficiency and antioxidant capacity (Ahmad et al., 2021 ). Exogenous MT pretreatment was able to effectively improve the root characteristics and enhance leaf photosynthesis and antioxidant capacity of tomato seedlings, therefore increasing its tolerance to drought stress (Altaf et al., 2022 ). In addition, with the intensifying heavy metal contamination of soils globally, the role of exogenous MT in enhancing heavy metal tolerance in plants has been increasingly valued (Arif et al., 2019 ). Ren et al. ( 2022 ) showed that exogenous MT could attenuate aluminum (Al) stress-induced growth inhibition by regulating maize carbon and nitrogen metabolism and maintaining its redox homeostasis. Meanwhile, exogenous MT pretreatment could enhance the tolerance of pepper to multiple heavy metal stresses (vanadium (V), chromium (Cr), Cd, and nickel (Ni)) by attenuating oxidative damage and decreasing heavy metal accumulation (Altaf et al., 2023 ). Ou et al. ( 2023 ) showed that exogenous MT treatment could reduce the accumulation of H 2 O 2 and malondialdehyde (MDA) in Platycladus orientalis caused by Cd stress, maintaining the stability of the membrane system and improving plant tolerance to Cd stress. Song et al. ( 2022 ) demonstrated that exogenous MT pretreatment could improve the tolerance of tobacco seedlings to Cd stress by promoting the development of glandular trichome and enhancing their photosynthetic and antioxidant capacities. Likewise, Cd stress causes severe growth and photosynthetic inhibition in tobacco seedlings, exogenous MT can promote the uptake of Cd 2+ in cell walls or vesicles of tobacco seedlings, thus reducing the amount of Cd 2+ accumulated in tobacco leaves. Besides, MT treatment also enhanced the antioxidant and photosynthetic capacity of tobacco, therefore enhancing the Cd stress tolerance of tobacco seedlings (Wang et al., 2019 ). H 2 O 2 and nitric oxide (NO) serve as two important signaling molecules, which are involved in plant development and stress response to abiotic stresses (Lee and Back, 2017 ; Mittler et al., 2022 ). Bagheri et al. ( 2019 ) found that H 2 O 2 treatment could increase plant height and root dry weight of pistachio seedlings. Exogenous NO promoted Kandelia obovata lateral root growth and morphology in a dose-dependent manner (Wei et al., 2022 ). It has also been found that H 2 O 2 plays a key role as a downstream signal of IAA in H 2 S-induced cold tolerance in cucumber seedlings (Zhang et al., 2021b ). Under heat stress, exogenous H 2 O 2 pretreatment enhanced antioxidant enzyme activities and attenuated oxidative damage caused by heat stress in perennial ryegrass, indicating that H 2 O 2 acts as a signaling molecule that triggers a protective metabolic response to heat stress in plants (Wang et al., 2014 ). Liao et al. ( 2012 ) indicated that NO and H 2 O 2 play key roles in the development of adventitious roots of marigold under drought stress, and NO at the appropriate concentration protects the ultrastructure of chloroplasts, improves the photosynthetic performance of leaves, and mitigates the negative effects of drought stress on the accumulation of sugars and nitrogen in the marigold explants. Liu et al. ( 2021 ) also noted that NO and H 2 O 2 can participate synergistically or antagonistically in a variety of biological processes, such as allergic response, stomatal movement, and abiotic stress response, which fully confirmed the close interaction between NO and H 2 O 2 in the regulation of plant growth and development and stress response. Interestingly, MT usually interacts with other signaling molecules in alleviating abiotic stress injury in plants. It has been reported that exogenous MT in alleviating chilling injury of cucumber seedlings, NO plays a key role as its downstream signaling molecule (Feng et al., 2021 ). Arora and Bhatla ( 2017 ) confirmed that MT may crosstalk with NO to maintain redox homeostasis in sunflower under salt stress, thereby relieving the oxidative damage caused by salt stress. Exogenous MT can induce local and systemic defenses against oxidative stress in cucumber seedlings, and the role of H 2 O 2 is crucial in this process (Li et al., 2016 ). Chen et al. ( 2018 ) have also found that exogenous MT requires the involvement of H 2 O 2 in the promotion of lateral root growth in alfalfa seedlings. Cucumber ( Cucumis sativus L.) is an important economic crop. It has been shown that cucumber is very sensitive to Cd toxicity during the seedling stage, and the high concentration of Cd can cause serious damage to its growth and development (Yang et al., 2023 ). At the same time, a large number of studies have also confirmed that exogenous MT treatments could effectively enhance plant tolerance to abiotic stresses such as salinity, high temperature, and heavy metal stresses, and that H 2 O 2 and NO play a key role in this process (Pardo-Hernández et al., 2020 ; Zhao et al., 2021 ; Zhu et al., 2019 ). However, there are few studies reported on the role of exogenous MT in alleviating the toxicity of Cd and enhancing the tolerance to Cd in cucumber seedlings. This study aimed to investigate the physiological and molecular mechanisms of exogenous MT in alleviating Cd stress injury in cucumber seedlings, and further to elucidate whether H 2 O 2 and NO signaling are involved in this process. 2. Materials and methods 2.1 Plant materials and stress treatments In this study, “Xinchun No. 4” cucumber was used as the experimental material (purchased from Kerun Seed Company Limited, Shandong, China). Healthy and full seeds were sown in pots with vermiculite and incubated. The growth chamber of seedlings was maintained at (25 ± 1) °C on a 16 h light/8 h dark. The maximum photosynthetic photon flux density (PPFD) was approximately 150 µmol·m − 2 ·s − 1 . After the cotyledons were fully expanded, cucumber seedlings of uniform growth were transferred to triangular vials containing nutrient solution for hydroponics. After the second true leaf of the cucumber is fully expanded the following treatments are performed: 1) CK: spray dH 2 O on the cucumber leaves every 12 h for 3 days (as “CK” of RNA-Seq); 2) MT treatment: spray 150 µM MT on the cucumber leaves every 12 h for 3 days; 3) Cadmium (Cd) stress treatment: after 3 days of dH 2 O pretreatment, seedlings were transferred to a Cd 2+ concentration of 150 µM in nutrient solution for 3 days (as treatments “D” of RNA-Seq). 4) MT + Cd treatment: foliar spraying of 150 µM MT every 12 h, after 3 days of spraying Cd stress treatment for 3 days (as treatments “MD” of RNA-Seq). 5) MT + Cd + DPI (NADPH oxidase inhibitor)/DMTU (H 2 O 2 scavenger)/cPTIO (NO scavenger)/L-NAME (NOS activity inhibitor)/tungstate (NR activity inhibitor) treatment: Foliar sprays of 150 µM MT were applied every 12 h. After 60 h, 100 µM DPI/5 mM DMTU/100 µM cPTIO/100 µM L-NAME/100 µM tungstate were sprayed, respectively. 12 h after the treatment was followed by Cd stress treatment for 3 days. After the treatment, the second true leaf was taken for the determination of physiological indices and RNA extraction. 2.2 Experimental Methods 2.2.1 Histochemical staining Diaminobenzidine (DAB) and Nitrotetrazolium Blue chloride (NBT) histochemical staining were used to detect H 2 O 2 and O 2 . − , respectively. Determination of H 2 O 2 : Cucumber leaves were placed in 1 mg/mL DAB solution and reacted for about 6 h. After brown spots appeared on the leaves, they were decolorized by boiling in decolorizing solution (95% ethanol) until chlorophyll was completely removed (Jambunathan, 2010 ). Determination of O 2 . − : Cucumber leaves were placed in 0.5 mg/mL NBT solution and reacted with light at 25°C for about 6 h. When dark blue spots appeared on the leaves, the color was decolorized by boiling in decolorizing solution (95% ethanol) (Kim et al., 2010 ). Finally, pictures were taken with a digital camera (Nikon Japan). 2.2.2 Determination of chlorophyll fluorescence parameters and photosynthetic pigment content Chlorophyll fluorescence parameters were determined using a proprietary portable photosynthesis-fluorescence measurement system (GFS-3000, Germany). After 30 min of dark adaptation of cucumber seedlings, the following chlorophyll fluorescence parameters were determined: Fv/Fm (PSII photochemical maximum quantum yield), Fv/Fo (PSII potential activity), Y(II) (effective quantum yield), Y(NPQ) (nonphotochemical quenching coefficient), qP (photochemical quenching coefficient), and ETR (photosynthetic electron transfer rate). The content of photosynthetic pigments in cucumber leaves was determined by the method of (Lin et al., 2021 ). Weigh 0.1 g cucumber leaves, soak in 95% ethanol, leave in the dark until the leaves turn white, take the supernatant and determine the absorbance values at 470 nm, 649 nm and 665 nm, respectively. 2.2.3 Determination of antioxidant enzyme activity The sample was taken 0.1 g, homogenized in 1 mL of pre-cooled extraction solution (50 mM PBS, pH 7.8, containing 0.1% PVP and 1 mM EDTA-Na 2 ), centrifuged at 4°C, 12000×g for 30 min, and the supernatant was taken as the crude enzyme solution. The SOD, CAT and POD activities in the enzyme solution were determined by the method of (Zhang et al., 2020 ). 2.2.4 transcriptome sequencing Total RNA of cucumber was extracted using RNAprep Pure Plant Kit (TIANGEN), and RNA sequencing of the samples was performed by Illumina HiSeq 2000 (Illumina, USA) platform. The sequencing was analyzed by Shanghai Ouyi Company. The raw data in Fastq format is processed using the Trimomatic program to obtain clean data. Clean data was calculated based on Q30, GC content, and repeat sequence levels, and RPKM (fragments per kilobase of transcript per million mapped reads) values were used to normalize gene expression levels to determine DEGs. Significance of DEGs was determined using FDR < 0.05 as the P-value threshold. Gene ontology (GO) enrichment analysis was performed according to the definition of DEGs of the GOseq R package. Q < 0.05 of GO terms were considered significantly enriched. Enrichment statistics of DEGs in the KEGG (Kyoto Encyclopedia of Genes and Genomics) pathway were performed using KOBAS software. 2.2.5 Quantitative real-time quantitative PCR (qRT-PCR) detection Total RNA from cucumber leaves was extracted using plant RNA extraction kit (MiniBEST plant RNA extraction kit), reverse transcription was performed according to the instructions of PrimeScript TM RT kit (Accurate Biotechnology, Hunan, China), and Takara TB Green ™ Premix Ex Taq™ II (Accurate Biotechnology, Hunan, China) was used for qRT-PCR analysis. The primers used in this study are listed in Tab. S1. 2.2.6 statistical analysis SPSS 25 was used for data processing and statistical analysis and Tukey's test was used for significance analysis with a significant difference level of P < 0.05. Graphs were prepared using Origin 2023 and TBtools software and data were expressed as mean ± standard error (SE). 3. Results 3.1 Effects of exogenous MT on the growth of cucumber seedlings under Cd stress Compared with CK, Cd treated cucumber seedling leaves showed Cd chelation spots, accompanied by yellowing phenomenon, and root Browning was also obvious. Compared with Cd treatment, MT + Cd treatment alleviated the toxic effects caused by Cd stress on cucumber seedlings. MT + Cd + DPI/DMTU and MT + Cd + cPTIO/L-NAME/tungstate treatments resulted in intensified yellowing of cucumber leaves, increased Cd chelating spotting, and browning of the root compared with MT + Cd treatment (Fig. 1 ). This result suggests that H 2 O 2 and NO play an important role in the mitigation of Cd stress in cucumber seedlings by MT. 3.2 Effects of exogenous MT on antioxidant system of cucumber seedlings under Cd stress Cd treatment increased H 2 O 2 and O 2 . − content in cucumber leaves compared with CK. MT + Cd treatment reduced the content of H 2 O 2 and O 2 . − compared with Cd treatment (Fig. 2 ). It indicated that exogenous MT pretreatment could reduce the accumulation of ROS in cucumber leaves. Excess ROS production usually activates the antioxidant defense system in plants. Therefore, the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and their gene expression levels were further examined in cucumber leaves. As shown in Fig. 3 , MT + Cd treatment significantly up-regulated the gene expression levels of SOD , POD , and CAT , as well as enhanced the activities of SOD, CAT, and POD, compared with Cd treatment. MT + Cd + DPI/DMTU and MT + Cd + cPTIO/L-NAME/tungstate treatments inhibited the activities and gene expression levels of these antioxidant enzymes compared to MT + Cd treatment. These results suggest that H 2 O 2 and NO play vital roles in exogenous MT to enhance antioxidant defense of cucumber seedlings under Cd stress. 3.3 Effects of exogenous MT on photosynthesis of cucumber seedlings under Cd stress As shown in Fig. 4 , chlorophyll a, carotenoids and total chlorophyll contents decreased by 36.8%, 36.8% and 27.3%, respectively, under Cd stress compared with CK. Whereas, exogenous MT treatment increased the content of photosynthetic pigments in cucumber leaves compared to Cd treatment. MT + Cd + DPI/DMTU and MT + Cd + cPTIO/L-NAME/tungstate treatments all decreased the content of photosynthetic pigments in cucumber leaves compared to MT + Cd treatment. Cd treatment reduced the chlorophyll fluorescence parameters (Fv/Fm, Fv/Fo, Y(II), Y(NPQ), qP and ETR) of cucumber seedlings. Compared with the Cd treatment, MT + Cd treatment increased the values of Fv/Fm, Fv/Fo, Y(II), Y(NPQ), qP and ETR of cucumber seedlings by 27.8%, 30%, 47.9%, 24%, 31% and 28.9%, respectively. Both MT + Cd + DPI/DMTU and MT + Cd + cPTIO/L-NAME/tungstate treatments resulted in decreasing chlorophyll fluorescence parameters of cucumber seedlings to different extents compared to MT + Cd treatment (Fig. 5 ). These results indicate that exogenous MT can increase the content of photosynthetic pigments in cucumber leaves and up-regulate the chlorophyll fluorescence parameters under Cd stress, and this process is regulated by H 2 O 2 and NO. 3.4 Effects of exogenous MT on WRKY transcription factor gene expression in cucumber seedlings under cadmium stress WRKY transcription factors are widely involved in plant responses to various abiotic stresses. As shown in Fig. 6 , Cd treatment significantly up-regulated the relative expression levels of WRKY33 , WRKY21 , WRKY38 and WRKY60 in cucumber seedlings compared with CK. MT + Cd treatment up-regulated the expression levels of WRKY33 , WRKY21 , WRKY38 , and WRKY60 by 47.5%, 180%, 150%, and 53.8%, respectively, compared with Cd treatment. However, compared with MT + Cd treatment, MT + Cd + DPI/DMTU and MT + Cd + cPTIO/L-NAME/tungstate treatments suppressed the expression levels of WRKY transcription factors. The above results indicated that exogenous MT pretreatment could induce the expression of WRKY transcription factors in cucumber seedlings under Cd stress, and H 2 O 2 and NO were involved in this regulatory process. 3.7 RNA sequencing and data analysis By constructing RNA-seq libraries of CK, D (Cd treatment) and MD (MT + Cd treatment) of cucumber seedlings and sequencing, a total of 65.38 Gb of clean data was obtained. The effective data volume of each sample was distributed in the range of 6.9–7.51 Gb, and the Q30 bases were distributed in the range of 90.69–94.22%, with an average GC content of 44.68% (Table. S2). The number of reads per sample compared to the reference genome was 96.52 to 98.76%. The number of reads compared to the reference genome was about 96.49%. The multiple mapped reads in each library were further analyzed, and a total of 4.38M to 4.84M Unique mapped reads were obtained for subsequent analysis, and the distribution of unique reads with positive/negative strands of the genome in each library was counted (Table. S3). Compared with CK, Cd (D) treatment up-regulated 2904 genes and down-regulated 2361 genes; MT + Cd (MD) treatment up-regulated 2025 genes and down-regulated 1787 genes. Compared with D treatment, MD treatment up-regulated 287 genes and down-regulated 1012 genes (Fig. 7 ). The above results indicated that exogenous MT could affect the gene expression profiles of cucumber seedlings under Cd stress. 3.8 GO classification and KEGG pathway analysis The result of GO enrichment analysis of differentially expressed genes (DEGs) in different treatment groups showed (Fig. 8 ) that DEGs were mainly enriched in biological processes such as toxin catabolic processes, photosynthesis, ionic homeostasis, stimulus response and hormone signaling pathways. In cellular components, DEGs are enriched in organelles and cell parts. In addition, they are enriched in cell membranes and membrane fractions. In molecular functions, DEGs are mainly enriched in transmembrane transporters, DNA-binding transcription factors, Cd 2+ binding, ribosomal structural components, and glutathione transferase activity. The KEGG enrichment analysis showed that numerous DEGs were enriched in photosynthesis, starch and sucrose metabolism, glutathione metabolism, and phytohormone signaling pathways, indicating that exogenous MT could enhance photosynthesis and antioxidant capacity, as well as activate phytohormone signaling pathways as a means of regulating the tolerance of cucumber seedlings to Cd stress. In addition, some DEGs were enriched in various metabolic and biosynthetic processes, including glycan, flavonoid biosynthesis, and tyrosine metabolic pathways. This indicates that MT can also regulate the growth of cucumber seedlings under Cd stress by influencing the energy metabolic processes in plants (Fig. 9 ). 3.9 DEGs related to the photosynthesis pathway The analysis of photosynthesis-related DEGs revealed that compared with CK treatment, Cd (D) treatment down-regulated the expression levels of most photosynthesis-related genes, such as PsbW (LOC101203467), PsbY (LOC101205582, LOC101205949), PsaK (LOC101209832), PsaN (LOC101204396), PsaF (LOC101215480), and LHCII (LOC101222687, LOC101203342, LOC101202993, LOC101207278), etc. Compared with Cd (D) treatment, MT + Cd (MD) treatment significantly up-regulated the expression levels of PsbW, PsbY, PsaK, PsaN, PsaF , and LHC II genes. These results indicated that exogenous MT treatment could restore the Cd-induced inhibition of photosynthesis-related gene expression and enhance photosynthesis in cucumber seedlings (Fig. 10 , Tab. S4). 3.10 DEGs related to antioxidant system To further explore the effects of exogenous MT on the antioxidant system of cucumber seedlings under Cd stress, the expression levels of DEGs related to the antioxidant system were analyzed under different treatment groups. Compared with Cd (D) treatment, MT + Cd (MD) treatment up-regulated the expression levels of DEGs encoding NADPH oxidase (LOC101206498, LOC101212879, LOC101217969 and LOC101218616), L-ascorbate oxidase (LOC101206777, LOC101206941) and polyamine oxidase (LOC101213852) and other antioxidant system related genes expression levels (Fig. 11 ). This indicates that exogenous MT treatment can enhance the tolerance of cucumber seedlings to Cd-induced oxidative stress by up-regulating the expression levels of genes encoding genes related to the antioxidant system of cucumber seedlings under Cd stress. 3.11 DEGs related to heavy metal transporter proteins Heavy metals cause toxicity to plants, but plants can also resist heavy metal toxicity by triggering avoidance and tolerance mechanisms. Heavy metal-responsive genes and transporter proteins play a pivotal role in this process. The analysis of DEGs revealed that compared with Cd (D) treatment, MT + Cd (MD) treatment down-regulated the expression levels of Nramp1 (LOC101211618), Nramp3 (LOC101211711), IRT (LOC101204028), CAX2 , HAM5.1 and HAM5.2 , and up-regulated the expression levels of genes encoding ABC transporter protein-related genes (LOC101210649, LOC101212585, LOC101214695, and LOC101219742) (Fig. 12 ). These results indicate that exogenous MT can down-regulate the expression of genes encoding cadmium transporter proteins to limit the uptake of Cd 2+ in cucumber seedlings. Meanwhile, it can also reduce Cd 2+ transport in plants by up-regulating the expression level of genes encoding cadmium chelate transporter proteins. Enhancing the tolerance of cucumber seedlings to Cd stress. 3.12 DEGs related to hormone metabolism As shown in Fig. 13 , the majority of the DEGs related to auxin (LOC101203821, LOC101204067, LOC101208132), brassinolide (LOC101213466, LOC101216594, LOC101217020), and jasmonic acid (LOC101212037) were up-regulated after MT + Cd (MD) treatment compared to Cd (D)treatment. However, DEGs related to ethylene (LOC101206564, LOC101210326) and abscisic acid (LOC101210966, LOC101212875) were down-regulated. This indicates that exogenous MT does not work alone in alleviating the Cd stress injury in cucumber seedlings, which is alleviated by forming a regulatory network with various other phytohormones. 3.13 DEGs related to transcription factors (TFs) TFs are proteins with the ability to bind DNA in a sequence-specific manner and regulate transcription, which can influence plant responses to biotic and abiotic stresses by controlling downstream responses through regulation of the transcription of target genes. To investigate the role of TFs in exogenous MT in alleviating Cd stress injury in cucumber seedlings, TFs-related DEGs were analyzed in this study. The results showed that WRKY (LOC101205904, LOC101206605, LOC101212435 and LOC101216931), bHLH (LOC101203107, LOC101203949, LOC101203971 and LOC101204984), MYB (LOC101203440, LOC101203786, and LOC101205447), PIF (LOC101205993, LOC101206441), and bZIP (LOC101214116, LOC101220715) were all involved in MT mitigation of Cd stress injury in cucumber seedlings. In addition, MT + Cd (MD) treatment up-regulated the expression levels of TFs-associated DEGs compared with Cd (D) treatment (Fig. 14 ). The above results suggest that TFs play a key role in the alleviation of Cd stress in cucumber seedlings by exogenous MT. 3.15 Verification of RNA-Seq by qRT-PCR In this study, 6 DEGs from RNA-seq were selected for qRT-PCR verification. The results showed a similar trend between the qRT-PCR analysis results and the RNA-Seq analysis, with a correlation of more than 90% (Fig. 15 ). This indicated that the RNA-Seq results were reliable. 4. Discussion Cd is a harmful non-essential element for plants. As one of the common environmental stresses, Cd stress severely disrupts the growth and development process of plants (Huybrechts et al., 2019 ). MT, as an important phytohormone, is actively involved in regulating plant responses to various abiotic stresses (Arnao and Hernández-Ruiz, 2015 ; Shi et al., 2016 ). Exogenous MT can alleviate Cd injury to Platycladus orientalis seedlings by scavenging ROS and maintaining membrane stability (Ou et al., 2023 ). In naked oat, exogenous MT could enhance the tolerance of oat seedlings to Cd stress by decreasing the contents of H 2 O 2 , O 2 . − , and MDA and increasing the activities of SOD, POD, and CAT (Wang et al., 2022a ). In this study, we found that exogenous MT pretreatment up-regulated the gene expression levels of SOD , POD , and CAT , enhanced SOD, POD, and CAT activities (Fig. 3 ), and decreased H 2 O 2 and O 2 . − content under Cd stress (Fig. 2 ). The RNA-seq results indicated that exogenous MT could upregulate the expression levels of genes encoding antioxidant system-related genes in cucumber seedlings with different degrees (Fig. 12 ). These indicate that under Cd stress, exogenous MT can improve plant antioxidant defense capacity by inducing the expression levels of genes related to antioxidant system, thereby reducing the excessive accumulation of ROS and enhancing the tolerance of cucumber seedlings to Cd. As we all know, Cd stress causes reduction of chlorophyll content and limits the synthesis of photosynthetic pigments in plants, which in return affects photosynthesis (Haider et al., 2021 ). It was found that exogenous MT significantly increased the content of photosynthetic pigments and enhanced the photosynthetic capacity of tea ( Camellia sinensis ) under Cd stress (Tan et al., 2022 ). In addition, exogenous MT pretreatment was able to alleviate the photosynthetic inhibition caused by low temperature (Wang et al., 2020 ) and high temperature stress (Jahan et al., 2021 ). In this study, we showed that Cd stress caused a decrease in photosynthetic pigment content in the leaves of cucumber seedlings, but exogenous MT increased chlorophyll a, carotenoids and total chlorophyll content (Fig. 4 ). It has been found that chlorophyll fluorescence parameters correlate strongly with plant survival after environmental stress, so it is considered a reliable indicator of response to stress intensity (Baker and Rosenqvist, 2004). In this study, chlorophyll fluorescence parameters (Fv/Fm, Fv/Fo, Y(II), Y(NPQ), qP, and ETR) were down-regulated in cucumber seedlings under Cd stress, and exogenous MT pretreatment significantly improved adverse effects caused by Cd stress on cucumber seedlings by up-regulating the levels of each chlorophyll fluorescence parameter (Fig. 5 ). RNA-seq results revealed that exogenous MT pretreatment up-regulated the expression of Psa N , Psa F and Psa K in photosystem I under Cd stress. Meanwhile, the expression levels of Psb W , Psb Y and LHC II in photosystem II were also significantly up-regulated. As important components of photosystem I, Psa N, Psa F and Psa K are involved in photosynthetic reactions in the photosynthetic electron transport chain (Schöttler et al., 2011 ). Bound to the periphery of photosystem II in higher plants is the major light-harvesting complex, LHC-II, which absorbs solar energy and delivers it to reaction centers, thereby facilitating photosynthesis (Chukhutsina et al., 2020 ). These results indicate that exogenous MT can up-regulate the levels of photosynthetic pigments and chlorophyll fluorescence parameters in the leaves of cucumber seedlings, as well as regulate the expression of photosynthesis-related genes to alleviate the photosynthetic inhibition caused by Cd stress, and enhance the photosynthesis of cucumber seedlings under Cd stress. In recent years, there have been numerous reports on the roles of H 2 O 2 and NO in plant defense against adversity stresses. Exogenous MT could delay ABA-induced senescence of tomato seedling leaves through H 2 O 2 -dependent Ca 2+ signaling (Guo et al., 2023 ). Zhang et al. ( 2022 ) found that NO is required for the ability of MT to increase nitrate tolerance in cucumber seedlings. In this study, we showed that Cd stress causes severe oxidative damage and photosynthetic inhibition in cucumber seedlings, and exogenous MT pretreatment can enhance the tolerance of cucumber seedlings to Cd by improving their antioxidant and photosynthetic capacities. However, the effects of MT were attenuated or even counteracted by the use of inhibitors and scavengers of H 2 O 2 or NO. These indicate that H 2 O 2 and NO are involved in regulating the exogenous MT to alleviate Cd stress injury in cucumber seedlings. A large number of studies have shown that MT is related to other known plant hormones in the regulation of plant stress tolerance(Arnao and Hernández-Ruiz, 2018 ). Exogenous MT treatment increased endogenous IAA content in maize seedlings under semi-arid conditions, which ensured plant survival under stress and increased maize yield (Ahmad et al., 2020 ). Under low-temperature stress, MT may act synergistically with jasmonic acid (JA) and IAA and antagonistically with abscisic acid (ABA) to regulate the tolerance of watermelon seedlings to low-temperature stress (Chang et al., 2020 ). Ding et al. ( 2022 ) showed that MT could synergize with JA to enhance low-temperature tolerance in tomato seedlings. Under drought and low-temperature stress, MT induced the synthesis of brassinolides (BRs) in perennial ryegrass leaves, and synergized with H 2 O 2 signaling to enhance the stress tolerance of plants (Fu et al., 2022 ). Jing et al. ( 2022 ) showed that MT could alleviate dark- and age-induced leaf senescence by activating the antioxidant system and IAA synthesis and signaling in cucumber seedlings while inhibiting ABA synthesis and signaling. When alfalfa seedlings were exposed to waterlogging stress, MT could attenuate plant damage by increasing polyamine (PA) content and decreasing ethylene (ETH) levels(Zhang et al., 2019 ). Consistent with previous findings, in this study, RNA-seq results showed that exogenous MT pretreatment significantly altered the expression levels of several phytohormones in cucumber seedlings under Cd stress. Among them, the related genes encoding hormones such as IAA, BRs and JA were up-regulated, while the genes related to ETH and ABA were down-regulated (Fig. 14 ). The above results indicated that exogenous MT did not act alone in alleviating the Cd stress injury in cucumber seedlings, but formed a regulatory network with other phytohormones to alleviate the stress injury suffered by cucumber seedlings. Transcription factors can transmit and amplify environmental stress signals by regulating the expression of their downstream stress-related genes (Vogel et al., 2005 ). It has been shown that bZIP transcription factors are involved in plant response processes to a variety of abiotic stresses, such as extreme temperature, water deficit, high osmotic pressure, and salinity (Li et al., 2020 ). Sapara et al. ( 2019 ) showed that the SbMYB15 transcription factor could mitigate Cd and nickel (Ni) stress damage in transgenic tobacco by limiting the uptake of heavy metal ions and regulating the antioxidant defense system. In addition, it has also been demonstrated that overexpression of transcription factors such as WRKY, bHLH, MYB, PIF, and bZIP can enhance stress tolerance in plants(Cai et al., 2018 ; Du et al., 2022 ; Qin et al., 2012 ; Zhao et al., 2018 ). Similar to previous studies, this study found that transcription factors such as WRKY, bHLH, MYB, PIF and bZIP were involved in the alleviation of Cd stress injury in cucumber seedlings by exogenous MT. The above results suggest that exogenous MT can be involved in regulating the tolerance of cucumber seedlings to Cd stress by up-regulating the expression levels of transcription factors. The plants have their own regulatory system to maintain the balance of metal ion concentration, and heavy metal transporter proteins are components of it, which are categorized into metal uptake proteins and metal efflux proteins, which play a crucial role in the uptake and tolerance of heavy metals in plants. Cd transport in plants is divided into two main parts, one part is Cd transporter proteins. For example, natural resistance and macrophage protein (NRAMP), zinc/iron transport protein (ZRT, IRT-like protein, ZIP), heavy metal ATPase (HMA) and cation exchanger (cation/proton antiporter, CAX). The other part is cadmium chelate transporter proteins, including yellow-stripe 1-like transporter (YSL) and ATP-binding cassette (ABC) (Yang et al., 2022 ). It has been shown when plants are exposed to Mn or Cd stress it leads to differential expression of three metal transporter genes ( HMA3, ABC15 and ATPase4 ), and that ABC transporter proteins play an important role in Mn and Cd detoxification (Fu et al., 2022 ). In this study, we found that exogenous MT pretreatment significantly down-regulated the expression levels of Nramp1, Nramp3, CAX2, HAM5.1 , and HAM5.2 . And the gene encoding cadmium chelate transporter protein ABC was up-regulated by MT. These results indicate that exogenous MT can limit the uptake of Cd 2+ of cucumber seedlings by down-regulating the expression of genes encoding cadmium transporter proteins and also reduce the transport of Cd 2+ in plants by up-regulating the expression level of genes encoding cadmium chelator transporter proteins. Thereby, the accumulation of Cd 2+ in the cucumber seedlings was reduced which in turn enhanced the tolerance of the cucumber seedlings to Cd stress. 5. Conclusion In this study, we used the cucumber "Xinchun No. 4" as the experimental material, combined with pharmacological experiments and transcriptome sequencing analysis, we revealed the molecular mechanism of exogenous MT-induced Cd stress tolerance in cucumber seedlings. The results showed that exogenous MT pretreatment could alleviate Cd stress injury by improving the antioxidant system and photosynthetic capacity of cucumber seedlings, and H 2 O 2 and NO also played important roles. Furthermore, phytohormones (IAA, BRs, JA, ABA, and ETH), various types of transcription factors (WRKY, bHLH, MYB, PIF, and bZIP), and heavy-metal transporter proteins (NRAMP, CAX, HAM, and ABC) all play an important role in the regulation of Cd stress tolerance in cucumber seedlings by MT. Altogether, these results provide new insights for further understanding of MT in improving plant Cd stress tolerance. Declarations CRediT authorship contribution statement Teng-Guo Zhang and Sheng Zheng conceived and designed the study; Xin Kang, Zi-Qi Pei and Ting-Ting Xu performed the bioinformatics analyses and the real-time quantitative PCR experiments; Cui-Yun Dong and Xue Bai helped to prepare figures and tables; Zi-Qi Pei wrote the manuscript; Teng-Guo Zhang, Juan Wang and Xin Kang reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Declaration of Competing Interest We declare that there is no conflict of interest between our papers and any organization or individual. Data availability Data will be made available on request. Funding sources This work was supported by the National Natural Science Foundation of China (32060711), Gansu Provincial Education and Science Technology Innovation Project (2022CXZX-357). References Ahmad, S., Cui, W., Kamran, M., Ahmad, I., Meng, X., Wu, X., Su, W., Javed, T., El-Serehy, H.A., Jia, Z., Han, Q., 2021. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3365346","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":236117327,"identity":"eb93809a-b60a-49c9-a0b3-cd8773b198b2","order_by":0,"name":"Xin Kang","email":"","orcid":"","institution":"Northwest Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Kang","suffix":""},{"id":236117328,"identity":"8dcda117-7532-498f-b710-5d9968a59097","order_by":1,"name":"Zi-Qi Pei","email":"","orcid":"","institution":"Northwest Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zi-Qi","middleName":"","lastName":"Pei","suffix":""},{"id":236117329,"identity":"d5f92389-7829-4009-ad38-b34811c09318","order_by":2,"name":"Ting-Ting Xu","email":"","orcid":"","institution":"Northwest Normal University","correspondingAuthor":false,"prefix":"","firstName":"Ting-Ting","middleName":"","lastName":"Xu","suffix":""},{"id":236117330,"identity":"83be0724-2ea0-43d3-8b97-6eae510a8085","order_by":3,"name":"Cui-Yun Dong","email":"","orcid":"","institution":"Northwest Normal University","correspondingAuthor":false,"prefix":"","firstName":"Cui-Yun","middleName":"","lastName":"Dong","suffix":""},{"id":236117331,"identity":"791bec95-5236-4b47-bd2d-02074db36073","order_by":4,"name":"Xue Bai","email":"","orcid":"","institution":"Northwest Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"Bai","suffix":""},{"id":236117332,"identity":"9243f86d-597d-4174-add7-3e84a0f87e3c","order_by":5,"name":"Juan Wang","email":"","orcid":"","institution":"Northwest Normal University","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Wang","suffix":""},{"id":236117333,"identity":"694df80c-1055-4ccc-8318-c27c66caf145","order_by":6,"name":"Sheng Zheng","email":"","orcid":"","institution":"Northwest Normal University","correspondingAuthor":false,"prefix":"","firstName":"Sheng","middleName":"","lastName":"Zheng","suffix":""},{"id":236117334,"identity":"501758d6-ca19-4f0c-a142-e49555426c08","order_by":7,"name":"Teng-Guo Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYDACCQglB+UyE6slgcGYdC2JDURrkZ/d/Ozh1x+16dslco9JMFRYJzawnz2AVwvjnGPmxjIJx3N3zshLk2A4k57YwJOXgFcLs0SCmbREwrHcDbdzzCQY2w4nNkjwGODVwiaR/g2kJd0ArOUfEVp4JHLMJD8k1CRAtDQQoUVCIqdMmiHtgOGG+2+MLYC2Gbfx5ODXIj8jfZvkD5s6eYMzZwxvfKixlu1nP4NfCwgw8zAchrASQL4jqB4IGH8w1BGjbhSMglEwCkYqAABpyUFqc4hqDAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-2166-0601","institution":"Northwest Normal University","correspondingAuthor":true,"prefix":"","firstName":"Teng-Guo","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2023-09-18 09:09:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3365346/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3365346/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11756-024-01670-0","type":"published","date":"2024-03-28T15:01:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":44047019,"identity":"1d3790f4-ddb2-4f7c-975f-3b37fa1ba3bb","added_by":"auto","created_at":"2023-10-03 22:57:20","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5640102,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of exogenous melatonin on the growth of cucumber seedlings under cadmium stress\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/7435b71148080f4b8104f444.jpeg"},{"id":44044612,"identity":"422facb4-11f1-4521-bcc0-d4701a1ff3eb","added_by":"auto","created_at":"2023-10-03 22:41:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":13847346,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of exogenous MT on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e.-\u003c/sup\u003e of cucumber seedlings under cadmium stress. A: DAB staining; B: NBT staining\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/9928f22e804c5c95369b997e.png"},{"id":44044606,"identity":"a8d152ce-0c11-45e3-80ec-c3775cbbcd8f","added_by":"auto","created_at":"2023-10-03 22:41:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4618954,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of exogenous MT on antioxidant enzyme activity and gene expression in cucumber seedlings under cadmium stress\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/31c5ba9b275ddf249f521242.png"},{"id":44044610,"identity":"8cf46dde-87ea-44f6-8eab-dbaf55209323","added_by":"auto","created_at":"2023-10-03 22:41:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2489703,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of exogenous melatonin on photosynthetic pigment content of cucumber seedlings under cadmium stress\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/4e948c94fbd969b04a5df7aa.png"},{"id":44045799,"identity":"87d95a8a-c981-4de3-8193-445adafa808d","added_by":"auto","created_at":"2023-10-03 22:49:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4218995,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of exogenous melatonin on chlorophyll fluorescence parameters of cucumber seedlings under cadmium stress\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/cffcf58692e5da3181d540b3.png"},{"id":44044609,"identity":"ad857b0d-e705-4973-8b35-2d64334f4ad9","added_by":"auto","created_at":"2023-10-03 22:41:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":208097,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of exogenous melatonin on WRKY transcription factor gene expression in cucumber seedlings under cadmium stress\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/c7475d59aa677ccea19f7ede.png"},{"id":44044624,"identity":"71102ab7-19b0-4145-9e21-9b5bef372726","added_by":"auto","created_at":"2023-10-03 22:41:20","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":7579911,"visible":true,"origin":"","legend":"\u003cp\u003eDEGs analyzed by RNA-seq. A: the number of DEGs; B: Venn diagram; C: volcano diagram\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/ab5d78685d6cffc70af0670d.png"},{"id":44044619,"identity":"7fe3eb4a-ca91-4a77-be54-c3db11e1091b","added_by":"auto","created_at":"2023-10-03 22:41:20","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":6620039,"visible":true,"origin":"","legend":"\u003cp\u003eGO classification of DEGs in D_vs_CK, MD_vs_CK and MD_vs_D\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/79c92410a798533cbda16e84.png"},{"id":44044615,"identity":"3f7df7ba-a318-46e8-a61e-50f89c1d10d5","added_by":"auto","created_at":"2023-10-03 22:41:20","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":6608529,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG pathway of cucumber seedlings DEGs in D_vs_CK , MD_vs_CK and MD_vs_D\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/07fa6a4551977704c9838a8a.png"},{"id":44044617,"identity":"3cff05be-68b5-4aaa-a36c-de318687ccf9","added_by":"auto","created_at":"2023-10-03 22:41:20","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2691319,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map of DEGs associated with Photosynthesis-related in MD_vs_D, MD_vs_CK and D_vs_CK.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/4eb342db12b20ad191c91a62.png"},{"id":44044614,"identity":"5cc2be85-3f74-4f31-9190-5e5f6963ccb4","added_by":"auto","created_at":"2023-10-03 22:41:20","extension":"jpeg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":229384,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map of DEGs associated with Membrane lipid peroxidation in MD_vs_D, MD_vs_CK and D_vs_CK.\u003c/p\u003e","description":"","filename":"floatimage11.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/8e9ca6090a3dd49aceff2bcb.jpeg"},{"id":44045800,"identity":"d9d768f7-6900-4597-abba-69de3629b3e8","added_by":"auto","created_at":"2023-10-03 22:49:20","extension":"jpeg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":297436,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map of DEGs associated with heavy metaltransporters in MD_vs_D, MD_vs_CK and D_vs_CK.\u003c/p\u003e","description":"","filename":"floatimage12.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/3efff245bb0d128f4764ac98.jpeg"},{"id":44044623,"identity":"ca175577-a146-4f2d-a06d-5db33584e96f","added_by":"auto","created_at":"2023-10-03 22:41:20","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":3761403,"visible":true,"origin":"","legend":"\u003cp\u003eHeat maps related to hormonal action in MD_vs_D, MD_vs_CK and D_vs_CK.\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/f02dcb7d0d7a20ee1464c12e.png"},{"id":44044622,"identity":"446b0f16-8f93-42b9-98ec-ddfdcb69c8ae","added_by":"auto","created_at":"2023-10-03 22:41:20","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":2556993,"visible":true,"origin":"","legend":"\u003cp\u003eHeat maps associated with transcription factor action in MD_vs_D, MD_vs_CK and D_vs_CK.\u003c/p\u003e","description":"","filename":"floatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/18bfd5b28df4dbf0cfdf7e08.png"},{"id":44044621,"identity":"d1bd549c-28fd-4cdb-b42a-167d77cface0","added_by":"auto","created_at":"2023-10-03 22:41:20","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":2807118,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative real-time PCR verification and correlation analysis with RNA-seq\u003c/p\u003e","description":"","filename":"floatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/719b142dd8fafb9a1c7e83c1.png"},{"id":53870110,"identity":"1ca30887-7a2f-4122-95e7-7374880b2613","added_by":"auto","created_at":"2024-04-01 15:13:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4627122,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/280ae590-7632-4568-8eaa-1834a53952d0.pdf"},{"id":44044605,"identity":"cc4d6eb4-d892-44a8-9b72-9ffb7dcc4edf","added_by":"auto","created_at":"2023-10-03 22:41:20","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16547,"visible":true,"origin":"","legend":"","description":"","filename":"renamed273c9.docx","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/eabcaaae71aff4b7c09e10fd.docx"},{"id":44045796,"identity":"3d1a9910-a8e1-4ef0-a4de-5f44e58ee003","added_by":"auto","created_at":"2023-10-03 22:49:20","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":22656,"visible":true,"origin":"","legend":"","description":"","filename":"renamed832b0.docx","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/d7a1976e1aa810b7192d4b4d.docx"},{"id":44047017,"identity":"de759e6c-3678-461e-b4e0-8a5371b68a11","added_by":"auto","created_at":"2023-10-03 22:57:20","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":18479,"visible":true,"origin":"","legend":"","description":"","filename":"renamed9188b.docx","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/ed827a9c978467b7f1ac2bcf.docx"},{"id":44045798,"identity":"affa16e7-5375-4501-bbb2-a9dcb256e149","added_by":"auto","created_at":"2023-10-03 22:49:20","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":18256,"visible":true,"origin":"","legend":"","description":"","filename":"renamed964bf.docx","url":"https://assets-eu.researchsquare.com/files/rs-3365346/v1/a7a378f11f926b47c9a45f8f.docx"}],"financialInterests":"","formattedTitle":"Exogenous melatonin enhanced cadmium stress tolerance of cucumber seedlings (Cucumis sativus L.)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn recent years, with the rapid development of industry and the excessive use of agricultural chemicals, the problem of heavy metal pollution of soil has become increasingly serious (Haider et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As a non-essential metal element for plant growth and development, cadmium (Cd) is easily absorbed by plants. Excessive accumulation of Cd\u003csup\u003e2+\u003c/sup\u003e can injure the structure and function of the plant membrane system and cause oxidative damage, which in reverse inhibits plant development and growth (Arif et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; He et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Huybrechts et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition, the accumulation of large amounts of Cd\u003csup\u003e2+\u003c/sup\u003e in plants can cause a variety of toxic symptoms, such as leaf yellowing, photosynthetic inhibition, root elongation inhibition, root tip browning, and biomass reduction, which can even lead to the death of the plant (Fan et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mohamed et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMelatonin (MT) is a multifunctional molecule that is ubiquitously found in various plants. Numerous literatures have reported that MT not only promotes plant growth and development (Arnao and Hern\u0026aacute;ndez-Ruiz, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Teng et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e) but it also plays an important role in enhancing abiotic stress tolerance in plants (Shi et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For example, Xiao et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) showed that low concentrations of MT could promote cotton seed germination by regulating hormone levels and antioxidant enzyme activities. Meanwhile, exogenous MT can also promote the elongation of Arabidopsis primary roots in an indole acetic acid (IAA)-dependent manner (Yang et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). When cucumber is suffering from low-temperature stress, exogenous MT can enhance the tolerance of cucumber seedlings to low-temperature stress by modulating its antioxidant system and photosynthesis(Zhang et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). Under salt stress, exogenous MT can enhance salt tolerance of maize seedlings by increasing their photosynthetic efficiency and antioxidant capacity (Ahmad et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Exogenous MT pretreatment was able to effectively improve the root characteristics and enhance leaf photosynthesis and antioxidant capacity of tomato seedlings, therefore increasing its tolerance to drought stress (Altaf et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition, with the intensifying heavy metal contamination of soils globally, the role of exogenous MT in enhancing heavy metal tolerance in plants has been increasingly valued (Arif et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Ren et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) showed that exogenous MT could attenuate aluminum (Al) stress-induced growth inhibition by regulating maize carbon and nitrogen metabolism and maintaining its redox homeostasis. Meanwhile, exogenous MT pretreatment could enhance the tolerance of pepper to multiple heavy metal stresses (vanadium (V), chromium (Cr), Cd, and nickel (Ni)) by attenuating oxidative damage and decreasing heavy metal accumulation (Altaf et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Ou et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) showed that exogenous MT treatment could reduce the accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and malondialdehyde (MDA) in \u003cem\u003ePlatycladus orientalis\u003c/em\u003e caused by Cd stress, maintaining the stability of the membrane system and improving plant tolerance to Cd stress. Song et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) demonstrated that exogenous MT pretreatment could improve the tolerance of tobacco seedlings to Cd stress by promoting the development of glandular trichome and enhancing their photosynthetic and antioxidant capacities. Likewise, Cd stress causes severe growth and photosynthetic inhibition in tobacco seedlings, exogenous MT can promote the uptake of Cd\u003csup\u003e2+\u003c/sup\u003e in cell walls or vesicles of tobacco seedlings, thus reducing the amount of Cd\u003csup\u003e2+\u003c/sup\u003e accumulated in tobacco leaves. Besides, MT treatment also enhanced the antioxidant and photosynthetic capacity of tobacco, therefore enhancing the Cd stress tolerance of tobacco seedlings (Wang et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and nitric oxide (NO) serve as two important signaling molecules, which are involved in plant development and stress response to abiotic stresses (Lee and Back, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mittler et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Bagheri et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) found that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment could increase plant height and root dry weight of pistachio seedlings. Exogenous NO promoted \u003cem\u003eKandelia obovata\u003c/em\u003e lateral root growth and morphology in a dose-dependent manner (Wei et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It has also been found that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e plays a key role as a downstream signal of IAA in H\u003csub\u003e2\u003c/sub\u003eS-induced cold tolerance in cucumber seedlings (Zhang et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). Under heat stress, exogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e pretreatment enhanced antioxidant enzyme activities and attenuated oxidative damage caused by heat stress in perennial ryegrass, indicating that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e acts as a signaling molecule that triggers a protective metabolic response to heat stress in plants (Wang et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Liao et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) indicated that NO and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e play key roles in the development of adventitious roots of marigold under drought stress, and NO at the appropriate concentration protects the ultrastructure of chloroplasts, improves the photosynthetic performance of leaves, and mitigates the negative effects of drought stress on the accumulation of sugars and nitrogen in the marigold explants. Liu et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) also noted that NO and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e can participate synergistically or antagonistically in a variety of biological processes, such as allergic response, stomatal movement, and abiotic stress response, which fully confirmed the close interaction between NO and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the regulation of plant growth and development and stress response.\u003c/p\u003e \u003cp\u003eInterestingly, MT usually interacts with other signaling molecules in alleviating abiotic stress injury in plants. It has been reported that exogenous MT in alleviating chilling injury of cucumber seedlings, NO plays a key role as its downstream signaling molecule (Feng et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Arora and Bhatla (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) confirmed that MT may crosstalk with NO to maintain redox homeostasis in sunflower under salt stress, thereby relieving the oxidative damage caused by salt stress. Exogenous MT can induce local and systemic defenses against oxidative stress in cucumber seedlings, and the role of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is crucial in this process (Li et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Chen et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) have also found that exogenous MT requires the involvement of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the promotion of lateral root growth in alfalfa seedlings.\u003c/p\u003e \u003cp\u003eCucumber (\u003cem\u003eCucumis sativus\u003c/em\u003e L.) is an important economic crop. It has been shown that cucumber is very sensitive to Cd toxicity during the seedling stage, and the high concentration of Cd can cause serious damage to its growth and development (Yang et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). At the same time, a large number of studies have also confirmed that exogenous MT treatments could effectively enhance plant tolerance to abiotic stresses such as salinity, high temperature, and heavy metal stresses, and that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NO play a key role in this process (Pardo-Hern\u0026aacute;ndez et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, there are few studies reported on the role of exogenous MT in alleviating the toxicity of Cd and enhancing the tolerance to Cd in cucumber seedlings. This study aimed to investigate the physiological and molecular mechanisms of exogenous MT in alleviating Cd stress injury in cucumber seedlings, and further to elucidate whether H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NO signaling are involved in this process.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant materials and stress treatments\u003c/h2\u003e \u003cp\u003eIn this study, \u0026ldquo;Xinchun No. 4\u0026rdquo; cucumber was used as the experimental material (purchased from Kerun Seed Company Limited, Shandong, China). Healthy and full seeds were sown in pots with vermiculite and incubated. The growth chamber of seedlings was maintained at (25\u0026thinsp;\u0026plusmn;\u0026thinsp;1) \u0026deg;C on a 16 h light/8 h dark. The maximum photosynthetic photon flux density (PPFD) was approximately 150 \u0026micro;mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. After the cotyledons were fully expanded, cucumber seedlings of uniform growth were transferred to triangular vials containing nutrient solution for hydroponics.\u003c/p\u003e \u003cp\u003eAfter the second true leaf of the cucumber is fully expanded the following treatments are performed: 1) CK: spray dH\u003csub\u003e2\u003c/sub\u003eO on the cucumber leaves every 12 h for 3 days (as \u0026ldquo;CK\u0026rdquo; of RNA-Seq); 2) MT treatment: spray 150 \u0026micro;M MT on the cucumber leaves every 12 h for 3 days; 3) Cadmium (Cd) stress treatment: after 3 days of dH\u003csub\u003e2\u003c/sub\u003eO pretreatment, seedlings were transferred to a Cd\u003csup\u003e2+\u003c/sup\u003e concentration of 150 \u0026micro;M in nutrient solution for 3 days (as treatments \u0026ldquo;D\u0026rdquo; of RNA-Seq). 4) MT\u0026thinsp;+\u0026thinsp;Cd treatment: foliar spraying of 150 \u0026micro;M MT every 12 h, after 3 days of spraying Cd stress treatment for 3 days (as treatments \u0026ldquo;MD\u0026rdquo; of RNA-Seq). 5) MT\u0026thinsp;+\u0026thinsp;Cd\u0026thinsp;+\u0026thinsp;DPI (NADPH oxidase inhibitor)/DMTU (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenger)/cPTIO (NO scavenger)/L-NAME (NOS activity inhibitor)/tungstate (NR activity inhibitor) treatment: Foliar sprays of 150 \u0026micro;M MT were applied every 12 h. After 60 h, 100 \u0026micro;M DPI/5 mM DMTU/100 \u0026micro;M cPTIO/100 \u0026micro;M L-NAME/100 \u0026micro;M tungstate were sprayed, respectively. 12 h after the treatment was followed by Cd stress treatment for 3 days. After the treatment, the second true leaf was taken for the determination of physiological indices and RNA extraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental Methods\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Histochemical staining\u003c/h2\u003e \u003cp\u003eDiaminobenzidine (DAB) and Nitrotetrazolium Blue chloride (NBT) histochemical staining were used to detect H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e.\u003csup\u003e\u0026minus;\u003c/sup\u003e, respectively. Determination of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e: Cucumber leaves were placed in 1 mg/mL DAB solution and reacted for about 6 h. After brown spots appeared on the leaves, they were decolorized by boiling in decolorizing solution (95% ethanol) until chlorophyll was completely removed (Jambunathan, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Determination of O\u003csub\u003e2\u003c/sub\u003e.\u003csup\u003e\u0026minus;\u003c/sup\u003e: Cucumber leaves were placed in 0.5 mg/mL NBT solution and reacted with light at 25\u0026deg;C for about 6 h. When dark blue spots appeared on the leaves, the color was decolorized by boiling in decolorizing solution (95% ethanol) (Kim et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Finally, pictures were taken with a digital camera (Nikon Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Determination of chlorophyll fluorescence parameters and photosynthetic pigment content\u003c/h2\u003e \u003cp\u003eChlorophyll fluorescence parameters were determined using a proprietary portable photosynthesis-fluorescence measurement system (GFS-3000, Germany). After 30 min of dark adaptation of cucumber seedlings, the following chlorophyll fluorescence parameters were determined: Fv/Fm (PSII photochemical maximum quantum yield), Fv/Fo (PSII potential activity), Y(II) (effective quantum yield), Y(NPQ) (nonphotochemical quenching coefficient), qP (photochemical quenching coefficient), and ETR (photosynthetic electron transfer rate).\u003c/p\u003e \u003cp\u003eThe content of photosynthetic pigments in cucumber leaves was determined by the method of (Lin et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Weigh 0.1 g cucumber leaves, soak in 95% ethanol, leave in the dark until the leaves turn white, take the supernatant and determine the absorbance values at 470 nm, 649 nm and 665 nm, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Determination of antioxidant enzyme activity\u003c/h2\u003e \u003cp\u003eThe sample was taken 0.1 g, homogenized in 1 mL of pre-cooled extraction solution (50 mM PBS, pH 7.8, containing 0.1% PVP and 1 mM EDTA-Na\u003csub\u003e2\u003c/sub\u003e), centrifuged at 4\u0026deg;C, 12000\u0026times;g for 30 min, and the supernatant was taken as the crude enzyme solution. The SOD, CAT and POD activities in the enzyme solution were determined by the method of (Zhang et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4 transcriptome sequencing\u003c/h2\u003e \u003cp\u003eTotal RNA of cucumber was extracted using RNAprep Pure Plant Kit (TIANGEN), and RNA sequencing of the samples was performed by Illumina HiSeq 2000 (Illumina, USA) platform. The sequencing was analyzed by Shanghai Ouyi Company. The raw data in Fastq format is processed using the Trimomatic program to obtain clean data. Clean data was calculated based on Q30, GC content, and repeat sequence levels, and RPKM (fragments per kilobase of transcript per million mapped reads) values were used to normalize gene expression levels to determine DEGs. Significance of DEGs was determined using FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 as the P-value threshold.\u003c/p\u003e \u003cp\u003eGene ontology (GO) enrichment analysis was performed according to the definition of DEGs of the GOseq R package. Q\u0026thinsp;\u0026lt;\u0026thinsp;0.05 of GO terms were considered significantly enriched. Enrichment statistics of DEGs in the KEGG (Kyoto Encyclopedia of Genes and Genomics) pathway were performed using KOBAS software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.5 Quantitative real-time quantitative PCR (qRT-PCR) detection\u003c/h2\u003e \u003cp\u003e Total RNA from cucumber leaves was extracted using plant RNA extraction kit (MiniBEST plant RNA extraction kit), reverse transcription was performed according to the instructions of PrimeScript TM RT kit (Accurate Biotechnology, Hunan, China), and Takara TB Green \u0026trade; Premix Ex Taq\u0026trade; II (Accurate Biotechnology, Hunan, China) was used for qRT-PCR analysis. The primers used in this study are listed in Tab. S1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.2.6 statistical analysis\u003c/h2\u003e \u003cp\u003eSPSS 25 was used for data processing and statistical analysis and Tukey's test was used for significance analysis with a significant difference level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Graphs were prepared using Origin 2023 and TBtools software and data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1 Effects of exogenous MT on the growth of cucumber seedlings under Cd stress\u003c/h2\u003e\n\u003cp\u003eCompared with CK, Cd treated cucumber seedling leaves showed Cd chelation spots, accompanied by yellowing phenomenon, and root Browning was also obvious. Compared with Cd treatment, MT\u0026thinsp;+\u0026thinsp;Cd treatment alleviated the toxic effects caused by Cd stress on cucumber seedlings. MT\u0026thinsp;+\u0026thinsp;Cd\u0026thinsp;+\u0026thinsp;DPI/DMTU and MT\u0026thinsp;+\u0026thinsp;Cd\u0026thinsp;+\u0026thinsp;cPTIO/L-NAME/tungstate treatments resulted in intensified yellowing of cucumber leaves, increased Cd chelating spotting, and browning of the root compared with MT\u0026thinsp;+\u0026thinsp;Cd treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). This result suggests that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NO play an important role in the mitigation of Cd stress in cucumber seedlings by MT.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2 Effects of exogenous MT on antioxidant system of cucumber seedlings under Cd stress\u003c/h2\u003e\n\u003cp\u003eCd treatment increased H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e.\u003csup\u003e\u0026minus;\u003c/sup\u003e content in cucumber leaves compared with CK. MT\u0026thinsp;+\u0026thinsp;Cd treatment reduced the content of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e.\u003csup\u003e\u0026minus;\u003c/sup\u003e compared with Cd treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). It indicated that exogenous MT pretreatment could reduce the accumulation of ROS in cucumber leaves.\u003c/p\u003e\n\u003cp\u003eExcess ROS production usually activates the antioxidant defense system in plants. Therefore, the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and their gene expression levels were further examined in cucumber leaves. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, MT\u0026thinsp;+\u0026thinsp;Cd treatment significantly up-regulated the gene expression levels of \u003cem\u003eSOD\u003c/em\u003e, \u003cem\u003ePOD\u003c/em\u003e, and \u003cem\u003eCAT\u003c/em\u003e, as well as enhanced the activities of SOD, CAT, and POD, compared with Cd treatment. MT\u0026thinsp;+\u0026thinsp;Cd\u0026thinsp;+\u0026thinsp;DPI/DMTU and MT\u0026thinsp;+\u0026thinsp;Cd\u0026thinsp;+\u0026thinsp;cPTIO/L-NAME/tungstate treatments inhibited the activities and gene expression levels of these antioxidant enzymes compared to MT\u0026thinsp;+\u0026thinsp;Cd treatment. These results suggest that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NO play vital roles in exogenous MT to enhance antioxidant defense of cucumber seedlings under Cd stress.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3 Effects of exogenous MT on photosynthesis of cucumber seedlings under Cd stress\u003c/h2\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, chlorophyll a, carotenoids and total chlorophyll contents decreased by 36.8%, 36.8% and 27.3%, respectively, under Cd stress compared with CK. Whereas, exogenous MT treatment increased the content of photosynthetic pigments in cucumber leaves compared to Cd treatment. MT\u0026thinsp;+\u0026thinsp;Cd\u0026thinsp;+\u0026thinsp;DPI/DMTU and MT\u0026thinsp;+\u0026thinsp;Cd\u0026thinsp;+\u0026thinsp;cPTIO/L-NAME/tungstate treatments all decreased the content of photosynthetic pigments in cucumber leaves compared to MT\u0026thinsp;+\u0026thinsp;Cd treatment.\u003c/p\u003e\n\u003cp\u003eCd treatment reduced the chlorophyll fluorescence parameters (Fv/Fm, Fv/Fo, Y(II), Y(NPQ), qP and ETR) of cucumber seedlings. Compared with the Cd treatment, MT\u0026thinsp;+\u0026thinsp;Cd treatment increased the values of Fv/Fm, Fv/Fo, Y(II), Y(NPQ), qP and ETR of cucumber seedlings by 27.8%, 30%, 47.9%, 24%, 31% and 28.9%, respectively. Both MT\u0026thinsp;+\u0026thinsp;Cd\u0026thinsp;+\u0026thinsp;DPI/DMTU and MT\u0026thinsp;+\u0026thinsp;Cd\u0026thinsp;+\u0026thinsp;cPTIO/L-NAME/tungstate treatments resulted in decreasing chlorophyll fluorescence parameters of cucumber seedlings to different extents compared to MT\u0026thinsp;+\u0026thinsp;Cd treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). These results indicate that exogenous MT can increase the content of photosynthetic pigments in cucumber leaves and up-regulate the chlorophyll fluorescence parameters under Cd stress, and this process is regulated by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NO.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Effects of exogenous MT on WRKY transcription factor gene expression in cucumber seedlings under cadmium stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWRKY transcription factors are widely involved in plant responses to various abiotic stresses. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, Cd treatment significantly up-regulated the relative expression levels of \u003cem\u003eWRKY33\u003c/em\u003e, \u003cem\u003eWRKY21\u003c/em\u003e, \u003cem\u003eWRKY38\u003c/em\u003e and \u003cem\u003eWRKY60\u003c/em\u003e in cucumber seedlings compared with CK. MT\u0026thinsp;+\u0026thinsp;Cd treatment up-regulated the expression levels of \u003cem\u003eWRKY33\u003c/em\u003e, \u003cem\u003eWRKY21\u003c/em\u003e, \u003cem\u003eWRKY38\u003c/em\u003e, and \u003cem\u003eWRKY60\u003c/em\u003e by 47.5%, 180%, 150%, and 53.8%, respectively, compared with Cd treatment. However, compared with MT\u0026thinsp;+\u0026thinsp;Cd treatment, MT\u0026thinsp;+\u0026thinsp;Cd\u0026thinsp;+\u0026thinsp;DPI/DMTU and MT\u0026thinsp;+\u0026thinsp;Cd\u0026thinsp;+\u0026thinsp;cPTIO/L-NAME/tungstate treatments suppressed the expression levels of WRKY transcription factors. The above results indicated that exogenous MT pretreatment could induce the expression of WRKY transcription factors in cucumber seedlings under Cd stress, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NO were involved in this regulatory process.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e3.7 RNA sequencing and data analysis\u003c/h2\u003e\n\u003cp\u003eBy constructing RNA-seq libraries of CK, D (Cd treatment) and MD (MT\u0026thinsp;+\u0026thinsp;Cd treatment) of cucumber seedlings and sequencing, a total of 65.38 Gb of clean data was obtained. The effective data volume of each sample was distributed in the range of 6.9\u0026ndash;7.51 Gb, and the Q30 bases were distributed in the range of 90.69\u0026ndash;94.22%, with an average GC content of 44.68% (Table. S2). The number of reads per sample compared to the reference genome was 96.52 to 98.76%. The number of reads compared to the reference genome was about 96.49%. The multiple mapped reads in each library were further analyzed, and a total of 4.38M to 4.84M Unique mapped reads were obtained for subsequent analysis, and the distribution of unique reads with positive/negative strands of the genome in each library was counted (Table. S3).\u003c/p\u003e\n\u003cp\u003eCompared with CK, Cd (D) treatment up-regulated 2904 genes and down-regulated 2361 genes; MT\u0026thinsp;+\u0026thinsp;Cd (MD) treatment up-regulated 2025 genes and down-regulated 1787 genes. Compared with D treatment, MD treatment up-regulated 287 genes and down-regulated 1012 genes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). The above results indicated that exogenous MT could affect the gene expression profiles of cucumber seedlings under Cd stress.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003e3.8 GO classification and KEGG pathway analysis\u003c/h2\u003e\n\u003cp\u003eThe result of GO enrichment analysis of differentially expressed genes (DEGs) in different treatment groups showed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e) that DEGs were mainly enriched in biological processes such as toxin catabolic processes, photosynthesis, ionic homeostasis, stimulus response and hormone signaling pathways. In cellular components, DEGs are enriched in organelles and cell parts. In addition, they are enriched in cell membranes and membrane fractions. In molecular functions, DEGs are mainly enriched in transmembrane transporters, DNA-binding transcription factors, Cd\u003csup\u003e2+\u003c/sup\u003e binding, ribosomal structural components, and glutathione transferase activity.\u003c/p\u003e\n\u003cp\u003eThe KEGG enrichment analysis showed that numerous DEGs were enriched in photosynthesis, starch and sucrose metabolism, glutathione metabolism, and phytohormone signaling pathways, indicating that exogenous MT could enhance photosynthesis and antioxidant capacity, as well as activate phytohormone signaling pathways as a means of regulating the tolerance of cucumber seedlings to Cd stress. In addition, some DEGs were enriched in various metabolic and biosynthetic processes, including glycan, flavonoid biosynthesis, and tyrosine metabolic pathways. This indicates that MT can also regulate the growth of cucumber seedlings under Cd stress by influencing the energy metabolic processes in plants (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003e3.9 DEGs related to the photosynthesis pathway\u003c/h2\u003e\n\u003cp\u003eThe analysis of photosynthesis-related DEGs revealed that compared with CK treatment, Cd (D) treatment down-regulated the expression levels of most photosynthesis-related genes, such as \u003cem\u003ePsbW\u003c/em\u003e (LOC101203467), \u003cem\u003ePsbY\u003c/em\u003e (LOC101205582, LOC101205949), \u003cem\u003ePsaK\u003c/em\u003e (LOC101209832), \u003cem\u003ePsaN\u003c/em\u003e (LOC101204396), \u003cem\u003ePsaF\u003c/em\u003e (LOC101215480), and \u003cem\u003eLHCII\u003c/em\u003e (LOC101222687, LOC101203342, LOC101202993, LOC101207278), etc. Compared with Cd (D) treatment, MT\u0026thinsp;+\u0026thinsp;Cd (MD) treatment significantly up-regulated the expression levels of \u003cem\u003ePsbW, PsbY, PsaK, PsaN, PsaF\u003c/em\u003e, and \u003cem\u003eLHC II\u003c/em\u003e genes. These results indicated that exogenous MT treatment could restore the Cd-induced inhibition of photosynthesis-related gene expression and enhance photosynthesis in cucumber seedlings (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e, Tab. S4).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003e3.10 DEGs related to antioxidant system\u003c/h2\u003e\n\u003cp\u003eTo further explore the effects of exogenous MT on the antioxidant system of cucumber seedlings under Cd stress, the expression levels of DEGs related to the antioxidant system were analyzed under different treatment groups. Compared with Cd (D) treatment, MT\u0026thinsp;+\u0026thinsp;Cd (MD) treatment up-regulated the expression levels of DEGs encoding NADPH oxidase (LOC101206498, LOC101212879, LOC101217969 and LOC101218616), L-ascorbate oxidase (LOC101206777, LOC101206941) and polyamine oxidase (LOC101213852) and other antioxidant system related genes expression levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e). This indicates that exogenous MT treatment can enhance the tolerance of cucumber seedlings to Cd-induced oxidative stress by up-regulating the expression levels of genes encoding genes related to the antioxidant system of cucumber seedlings under Cd stress.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003e3.11 DEGs related to heavy metal transporter proteins\u003c/h2\u003e\n\u003cp\u003eHeavy metals cause toxicity to plants, but plants can also resist heavy metal toxicity by triggering avoidance and tolerance mechanisms. Heavy metal-responsive genes and transporter proteins play a pivotal role in this process. The analysis of DEGs revealed that compared with Cd (D) treatment, MT\u0026thinsp;+\u0026thinsp;Cd (MD) treatment down-regulated the expression levels of \u003cem\u003eNramp1\u003c/em\u003e (LOC101211618), \u003cem\u003eNramp3\u003c/em\u003e (LOC101211711), \u003cem\u003eIRT\u003c/em\u003e (LOC101204028), \u003cem\u003eCAX2\u003c/em\u003e, \u003cem\u003eHAM5.1\u003c/em\u003e and \u003cem\u003eHAM5.2\u003c/em\u003e, and up-regulated the expression levels of genes encoding ABC transporter protein-related genes (LOC101210649, LOC101212585, LOC101214695, and LOC101219742) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e). These results indicate that exogenous MT can down-regulate the expression of genes encoding cadmium transporter proteins to limit the uptake of Cd\u003csup\u003e2+\u003c/sup\u003e in cucumber seedlings. Meanwhile, it can also reduce Cd\u003csup\u003e2+\u003c/sup\u003e transport in plants by up-regulating the expression level of genes encoding cadmium chelate transporter proteins. Enhancing the tolerance of cucumber seedlings to Cd stress.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003e3.12 DEGs related to hormone metabolism\u003c/h2\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e, the majority of the DEGs related to auxin (LOC101203821, LOC101204067, LOC101208132), brassinolide (LOC101213466, LOC101216594, LOC101217020), and jasmonic acid (LOC101212037) were up-regulated after MT\u0026thinsp;+\u0026thinsp;Cd (MD) treatment compared to Cd (D)treatment. However, DEGs related to ethylene (LOC101206564, LOC101210326) and abscisic acid (LOC101210966, LOC101212875) were down-regulated. This indicates that exogenous MT does not work alone in alleviating the Cd stress injury in cucumber seedlings, which is alleviated by forming a regulatory network with various other phytohormones.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003e3.13 DEGs related to transcription factors (TFs)\u003c/h2\u003e\n\u003cp\u003eTFs are proteins with the ability to bind DNA in a sequence-specific manner and regulate transcription, which can influence plant responses to biotic and abiotic stresses by controlling downstream responses through regulation of the transcription of target genes. To investigate the role of TFs in exogenous MT in alleviating Cd stress injury in cucumber seedlings, TFs-related DEGs were analyzed in this study. The results showed that WRKY (LOC101205904, LOC101206605, LOC101212435 and LOC101216931), bHLH (LOC101203107, LOC101203949, LOC101203971 and LOC101204984), MYB (LOC101203440, LOC101203786, and LOC101205447), PIF (LOC101205993, LOC101206441), and bZIP (LOC101214116, LOC101220715) were all involved in MT mitigation of Cd stress injury in cucumber seedlings. In addition, MT\u0026thinsp;+\u0026thinsp;Cd (MD) treatment up-regulated the expression levels of TFs-associated DEGs compared with Cd (D) treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003e). The above results suggest that TFs play a key role in the alleviation of Cd stress in cucumber seedlings by exogenous MT.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003e3.15 Verification of RNA-Seq by qRT-PCR\u003c/h2\u003e\n\u003cp\u003eIn this study, 6 DEGs from RNA-seq were selected for qRT-PCR verification. The results showed a similar trend between the qRT-PCR analysis results and the RNA-Seq analysis, with a correlation of more than 90% (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e15\u003c/span\u003e). This indicated that the RNA-Seq results were reliable.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCd is a harmful non-essential element for plants. As one of the common environmental stresses, Cd stress severely disrupts the growth and development process of plants (Huybrechts et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). MT, as an important phytohormone, is actively involved in regulating plant responses to various abiotic stresses (Arnao and Hern\u0026aacute;ndez-Ruiz, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Shi et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Exogenous MT can alleviate Cd injury to \u003cem\u003ePlatycladus orientalis\u003c/em\u003e seedlings by scavenging ROS and maintaining membrane stability (Ou et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In naked oat, exogenous MT could enhance the tolerance of oat seedlings to Cd stress by decreasing the contents of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, O\u003csub\u003e2\u003c/sub\u003e.\u003csup\u003e\u0026minus;\u003c/sup\u003e, and MDA and increasing the activities of SOD, POD, and CAT (Wang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). In this study, we found that exogenous MT pretreatment up-regulated the gene expression levels of \u003cem\u003eSOD\u003c/em\u003e, \u003cem\u003ePOD\u003c/em\u003e, and \u003cem\u003eCAT\u003c/em\u003e, enhanced SOD, POD, and CAT activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), and decreased H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e.\u003csup\u003e\u0026minus;\u003c/sup\u003e content under Cd stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The RNA-seq results indicated that exogenous MT could upregulate the expression levels of genes encoding antioxidant system-related genes in cucumber seedlings with different degrees (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). These indicate that under Cd stress, exogenous MT can improve plant antioxidant defense capacity by inducing the expression levels of genes related to antioxidant system, thereby reducing the excessive accumulation of ROS and enhancing the tolerance of cucumber seedlings to Cd.\u003c/p\u003e \u003cp\u003eAs we all know, Cd stress causes reduction of chlorophyll content and limits the synthesis of photosynthetic pigments in plants, which in return affects photosynthesis (Haider et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It was found that exogenous MT significantly increased the content of photosynthetic pigments and enhanced the photosynthetic capacity of tea (\u003cem\u003eCamellia sinensis\u003c/em\u003e) under Cd stress (Tan et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition, exogenous MT pretreatment was able to alleviate the photosynthetic inhibition caused by low temperature (Wang et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and high temperature stress (Jahan et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this study, we showed that Cd stress caused a decrease in photosynthetic pigment content in the leaves of cucumber seedlings, but exogenous MT increased chlorophyll a, carotenoids and total chlorophyll content (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). It has been found that chlorophyll fluorescence parameters correlate strongly with plant survival after environmental stress, so it is considered a reliable indicator of response to stress intensity (Baker and Rosenqvist, 2004). In this study, chlorophyll fluorescence parameters (Fv/Fm, Fv/Fo, Y(II), Y(NPQ), qP, and ETR) were down-regulated in cucumber seedlings under Cd stress, and exogenous MT pretreatment significantly improved adverse effects caused by Cd stress on cucumber seedlings by up-regulating the levels of each chlorophyll fluorescence parameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). RNA-seq results revealed that exogenous MT pretreatment up-regulated the expression of \u003cem\u003ePsa N\u003c/em\u003e, \u003cem\u003ePsa F\u003c/em\u003e and \u003cem\u003ePsa K\u003c/em\u003e in photosystem I under Cd stress. Meanwhile, the expression levels of \u003cem\u003ePsb W\u003c/em\u003e, \u003cem\u003ePsb Y\u003c/em\u003e and \u003cem\u003eLHC II\u003c/em\u003e in photosystem II were also significantly up-regulated. As important components of photosystem I, Psa N, Psa F and Psa K are involved in photosynthetic reactions in the photosynthetic electron transport chain (Sch\u0026ouml;ttler et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Bound to the periphery of photosystem II in higher plants is the major light-harvesting complex, LHC-II, which absorbs solar energy and delivers it to reaction centers, thereby facilitating photosynthesis (Chukhutsina et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These results indicate that exogenous MT can up-regulate the levels of photosynthetic pigments and chlorophyll fluorescence parameters in the leaves of cucumber seedlings, as well as regulate the expression of photosynthesis-related genes to alleviate the photosynthetic inhibition caused by Cd stress, and enhance the photosynthesis of cucumber seedlings under Cd stress.\u003c/p\u003e \u003cp\u003eIn recent years, there have been numerous reports on the roles of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NO in plant defense against adversity stresses. Exogenous MT could delay ABA-induced senescence of tomato seedling leaves through H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-dependent Ca\u003csup\u003e2+\u003c/sup\u003e signaling (Guo et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Zhang et al. (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) found that NO is required for the ability of MT to increase nitrate tolerance in cucumber seedlings. In this study, we showed that Cd stress causes severe oxidative damage and photosynthetic inhibition in cucumber seedlings, and exogenous MT pretreatment can enhance the tolerance of cucumber seedlings to Cd by improving their antioxidant and photosynthetic capacities. However, the effects of MT were attenuated or even counteracted by the use of inhibitors and scavengers of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e or NO. These indicate that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NO are involved in regulating the exogenous MT to alleviate Cd stress injury in cucumber seedlings.\u003c/p\u003e \u003cp\u003eA large number of studies have shown that MT is related to other known plant hormones in the regulation of plant stress tolerance(Arnao and Hern\u0026aacute;ndez-Ruiz, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Exogenous MT treatment increased endogenous IAA content in maize seedlings under semi-arid conditions, which ensured plant survival under stress and increased maize yield (Ahmad et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Under low-temperature stress, MT may act synergistically with jasmonic acid (JA) and IAA and antagonistically with abscisic acid (ABA) to regulate the tolerance of watermelon seedlings to low-temperature stress (Chang et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Ding et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) showed that MT could synergize with JA to enhance low-temperature tolerance in tomato seedlings. Under drought and low-temperature stress, MT induced the synthesis of brassinolides (BRs) in perennial ryegrass leaves, and synergized with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e signaling to enhance the stress tolerance of plants (Fu et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Jing et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) showed that MT could alleviate dark- and age-induced leaf senescence by activating the antioxidant system and IAA synthesis and signaling in cucumber seedlings while inhibiting ABA synthesis and signaling. When alfalfa seedlings were exposed to waterlogging stress, MT could attenuate plant damage by increasing polyamine (PA) content and decreasing ethylene (ETH) levels(Zhang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Consistent with previous findings, in this study, RNA-seq results showed that exogenous MT pretreatment significantly altered the expression levels of several phytohormones in cucumber seedlings under Cd stress. Among them, the related genes encoding hormones such as IAA, BRs and JA were up-regulated, while the genes related to ETH and ABA were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003e). The above results indicated that exogenous MT did not act alone in alleviating the Cd stress injury in cucumber seedlings, but formed a regulatory network with other phytohormones to alleviate the stress injury suffered by cucumber seedlings.\u003c/p\u003e \u003cp\u003eTranscription factors can transmit and amplify environmental stress signals by regulating the expression of their downstream stress-related genes (Vogel et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). It has been shown that bZIP transcription factors are involved in plant response processes to a variety of abiotic stresses, such as extreme temperature, water deficit, high osmotic pressure, and salinity (Li et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Sapara et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) showed that the SbMYB15 transcription factor could mitigate Cd and nickel (Ni) stress damage in transgenic tobacco by limiting the uptake of heavy metal ions and regulating the antioxidant defense system. In addition, it has also been demonstrated that overexpression of transcription factors such as WRKY, bHLH, MYB, PIF, and bZIP can enhance stress tolerance in plants(Cai et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Du et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Qin et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Similar to previous studies, this study found that transcription factors such as WRKY, bHLH, MYB, PIF and bZIP were involved in the alleviation of Cd stress injury in cucumber seedlings by exogenous MT. The above results suggest that exogenous MT can be involved in regulating the tolerance of cucumber seedlings to Cd stress by up-regulating the expression levels of transcription factors.\u003c/p\u003e \u003cp\u003eThe plants have their own regulatory system to maintain the balance of metal ion concentration, and heavy metal transporter proteins are components of it, which are categorized into metal uptake proteins and metal efflux proteins, which play a crucial role in the uptake and tolerance of heavy metals in plants. Cd transport in plants is divided into two main parts, one part is Cd transporter proteins. For example, natural resistance and macrophage protein (NRAMP), zinc/iron transport protein (ZRT, IRT-like protein, ZIP), heavy metal ATPase (HMA) and cation exchanger (cation/proton antiporter, CAX). The other part is cadmium chelate transporter proteins, including yellow-stripe 1-like transporter (YSL) and ATP-binding cassette (ABC) (Yang et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It has been shown when plants are exposed to Mn or Cd stress it leads to differential expression of three metal transporter genes (\u003cem\u003eHMA3, ABC15\u003c/em\u003e and \u003cem\u003eATPase4\u003c/em\u003e), and that ABC transporter proteins play an important role in Mn and Cd detoxification (Fu et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this study, we found that exogenous MT pretreatment significantly down-regulated the expression levels of \u003cem\u003eNramp1, Nramp3, CAX2, HAM5.1\u003c/em\u003e, and \u003cem\u003eHAM5.2\u003c/em\u003e. And the gene encoding cadmium chelate transporter protein ABC was up-regulated by MT. These results indicate that exogenous MT can limit the uptake of Cd\u003csup\u003e2+\u003c/sup\u003e of cucumber seedlings by down-regulating the expression of genes encoding cadmium transporter proteins and also reduce the transport of Cd\u003csup\u003e2+\u003c/sup\u003e in plants by up-regulating the expression level of genes encoding cadmium chelator transporter proteins. Thereby, the accumulation of Cd\u003csup\u003e2+\u003c/sup\u003e in the cucumber seedlings was reduced which in turn enhanced the tolerance of the cucumber seedlings to Cd stress.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, we used the cucumber \u0026quot;Xinchun No. 4\u0026quot; as the experimental material, combined with pharmacological experiments and transcriptome sequencing analysis, we revealed the molecular mechanism of exogenous MT-induced Cd stress tolerance in cucumber seedlings. The results showed that exogenous MT pretreatment could alleviate Cd stress injury by improving the antioxidant system and photosynthetic capacity of cucumber seedlings, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NO also played important roles. Furthermore, phytohormones (IAA, BRs, JA, ABA, and ETH), various types of transcription factors (WRKY, bHLH, MYB, PIF, and bZIP), and heavy-metal transporter proteins (NRAMP, CAX, HAM, and ABC) all play an important role in the regulation of Cd stress tolerance in cucumber seedlings by MT. Altogether, these results provide new insights for further understanding of MT in improving plant Cd stress tolerance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTeng-Guo Zhang\u003c/strong\u003e and \u003cstrong\u003eSheng Zheng\u003c/strong\u003e conceived and designed the study; \u003cstrong\u003eXin Kang, Zi-Qi Pei\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eTing-Ting Xu\u003c/strong\u003e performed the bioinformatics analyses and the real-time quantitative PCR experiments; \u003cstrong\u003eCui-Yun Dong\u003c/strong\u003e and \u003cstrong\u003eXue Bai\u003c/strong\u003e helped to prepare figures and tables; \u003cstrong\u003eZi-Qi Pei\u003c/strong\u003e wrote the manuscript; \u003cstrong\u003eTeng-Guo Zhang, Juan Wang\u003c/strong\u003e and \u003cstrong\u003eXin Kang\u003c/strong\u003e reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare that there is no conflict of interest between our papers and any organization or individual.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32060711), Gansu Provincial Education and Science Technology Innovation Project (2022CXZX-357).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhmad, S., Cui, W., Kamran, M., Ahmad, I., Meng, X., Wu, X., Su, W., Javed, T., El-Serehy, H.A., Jia, Z., Han, Q., 2021. 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Int J Mol Sci 20, 6200. https://doi.org/10.3390/ijms20246200.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Cucumber, Melatonin, Cadmium stress, RNA-seq","lastPublishedDoi":"10.21203/rs.3.rs-3365346/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3365346/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMelatonin (MT) is a phytohormone that can improve plant stress resistance by regulating physiological processes and gene expression. The present study investigated the role of exogenous MT in alleviating cadmium (Cd) stress in cucumber seedlings. The results showed that Cd stress inhibited the growth of cucumber seedlings and exogenous MT reversed adverse effects of Cd stress. Compared with Cd treatment, MT\u0026thinsp;+\u0026thinsp;Cd treatment enhanced antioxidant enzyme activities, suppressed ROS production and improved photosynthesis in cucumber seedlings. Further research showed that hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) and nitric oxide (NO) played important roles in MT enhanced Cd stress tolerance in cucumber seedlings. RNA-seq results indicated that MT was not only related to photosynthetic and antioxidant systems in alleviating Cd injury in cucumber seedlings, but also various phytohormones, heavy metal transporter proteins and transcription factors were also involved. In addition, we selected six differentially expressed genes for qRT-PCR validation, the verification results were consistent with the RNA-seq results. In summary, exogenous MT pretreatment can alleviate Cd toxicity by enhancing antioxidant defense capacity and photosynthetic efficiency of cucumber seedlings, both H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NO play important roles. In addition, various phytohormones, transcription factors and heavy metal transport proteins are also involved in this regulation of MT.\u003c/p\u003e","manuscriptTitle":"Exogenous melatonin enhanced cadmium stress tolerance of cucumber seedlings (Cucumis sativus L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-03 22:41:15","doi":"10.21203/rs.3.rs-3365346/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-09-27T05:31:16+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-09-26T10:51:57+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Biologia","date":"2023-09-21T09:39:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-09-19T08:29:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biologia","date":"2023-09-18T05:09:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"859466e4-c09d-4562-a125-abbc292f1073","owner":[],"postedDate":"October 3rd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-04-01T15:12:26+00:00","versionOfRecord":{"articleIdentity":"rs-3365346","link":"https://doi.org/10.1007/s11756-024-01670-0","journal":{"identity":"biologia","isVorOnly":false,"title":"Biologia"},"publishedOn":"2024-03-28 15:01:42","publishedOnDateReadable":"March 28th, 2024"},"versionCreatedAt":"2023-10-03 22:41:15","video":"","vorDoi":"10.1007/s11756-024-01670-0","vorDoiUrl":"https://doi.org/10.1007/s11756-024-01670-0","workflowStages":[]},"version":"v1","identity":"rs-3365346","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3365346","identity":"rs-3365346","version":["v1"]},"buildId":"J0_U0BvcaRcwD8yVFaRlm","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}